It is known that blood clots are formed through the action of a plasma component known as fibrin.
It is also known that materials which tend to dissolve fibrin (fibrinolytic materials) are effective in reducing, or eliminating, blood clots in the circulatory system where great harm can result from circulatory blockages which may be produced by undissolved blood clots.
Human blood contains plasminogen which is not an enzyme but an enzyme precursor. Certain activators convert plasminogen to plasmin which is capable of dissolving fibrin and breaking up clots. Plasmin is a proteolytic enzyme having a serine protease active site and having the capability of dissolving protein, including itself. Plasmin and many of its complexes are therefore unstable in storage in solution form without the addition of stabilizing materials, such as leupeptin. The proteolytic activity of plasmin and its complexes can produce adverse secondary effects when such materials are introduced into the bloodstream since proteolytic activity can destroy serum proteins, such as clotting and complement components.
Streptokinase, derived from streptococcus cultures, is an activator capable of converting human plasminogen to human plasmin. It is also capable of converting cat plasminogen to cat plasmin, but is relatively inactive for the conversion of other mammalian plasminogens.
When streptokinase is added to human plasminogen, its first action is to combine with some of the plasminogen in stoichiometric proportions to form a complex. The streptokinase-plasminogen complex then serves as a catalyst for conversion of the remaining plasminogen to plasmin. Streptokinase also combines stoichiometrically with plasmin to form a complex which is a catalyst for the conversion of plasminogen.
Streptokinase, as indicated above, is relatively inactive for the conversion of mammalian plasminogens other than human and cat plasminogens, and thus does not convert bovine plasminogen to bovine plasmin. On the other hand, the streptokinase-human plasminogen complex and the streptokinase-human plasmin complex are active in the conversion of bovine plasminogen to bovine plasmin.
It is known that plasmin may be split into a heavy (A) chain a light (B) chain by cleavage of the plasmin molecules at their interchain disulfide bonds (one or two disulfide bonds per molecule) and it is known that the heavy (A) chain and light (B) chain may be separated from each other. Rickli and Otavasky reported in Eur. J. Biochem. 59, 441-447 (1975) that the fractions may be separated by adsorption of the heavy (A) chain on a L-lysine-substituted polyacrylamide adsorbent with elution of the light (B) chain fraction. The authors do not report any properties of the eluted light (B) chain fraction, but our tests have shown that the light (B) chain fraction produced by their method, when complexed with streptokinase, produces a complex which is substantially inactive for the conversion of plasminogen to plasmin.
The heavy (A) fraction cannot produce streptokinase complex.
For safety reasons, therapeutic materials derived from human blood, including plasminogen and plasmin, cannot be injected into the bloodstream of a patient because such injection can transmit viral contaminants, such as the virus of hepatitis, to the bloodstream of the patient. The Food and Drug Administration of the United States requires that any material derived from human blood be heated at 60.degree. C for ten hours to inactivate any hepatitis virus before it will approve the material, or any derivative thereof, for sale as a therapeutic material to be injected into the bloodstream.
Subjection of a streptokinase-plasmin complex to the above-described heat treatment alters its character and renders it inactive for conversion of plasminogen to plasmin. Subjection of plasminogen to the above-described heat treatment before converting it to plasmin and before complexing with streptokinase also alters the character of the resulting complex. Although its streptokinase complex still retains activity for the conversion of plasminogen to plasmin, its protein component is so denatured by the heat treatment that antigenic and pyrogenic reactions may be produced when the complex is injected into the bloodstream of a patient.
For these reasons, streptokinase-plasmin complexes have not been utilized as a standard therapeutic treatment for dissolving blood clots in human patients.